Summary: The class of mammalian biologically active polypeptides called growth factors influence the proliferation, differentiation, motility, maintenance and apoptosis of target cells. The ErbB family of receptors which include the epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4 mediate the biological actions of a family of growth factors which are structurally related to EGF. The EGFR, ErbB2 and ErbB4 are protein tyrosine kinases, whereas ErbB3 has no known intrinsic enzymatic activity. Engagement of growth factor by the extracellular domain of an ErbB initiates a process of hetero- and homo-oligomerization of ErbBs, transphosphorylation of the ErbBs in the complex and subsequenct signaling. It is important to note that no known extracellular growth factor ligand for ErbB2 has been identified. Relatively little is known regarding the molecular mechanism of ErbB kinase activation and how specificity of signaling is conferred. To generate insight into ErbB signaling our laboratory is studying the enzymology of the ErbBs. To this end, we have generated epitope-tagged versions of each of the ErbB receptors to facilitate an in vitro determination of the enzymatic parameters towards specific substrates. Synthetic peptide substrates corresponding to specific phosphorylation sites within the COOH-terminal regions of the ErbBs have been prepared for this analysis. In addition, mutant ErbBs are being generated in which tyrosine to phenylalanine mutations have been made at known transphosphorylation sites. These mutants will be used in intact cells to test various hypotheses which arise from the in vitro enzymatic studies. An additional area of interest involves the role of the EGFR kinase activity in EGF-dependent signaling. Gene targeting and inactivation of the murine EGFR results in strain-dependent phenotypes that range from death in utero to postnatal abnormalities in skin, kidney, lung, gastrointestinal tract, and brain. However, recent work from our lab has demonstrated that some EGF-dependent signals, such as activation of MAP kinase and the pro-survival kinase Akt, do not require the EGFR kinase activity in cells which also express ErbB2 (Deb et al., 2001). Since EGFR and ErbB2 are almost always coexpressed in fibroblasts and epithelial cells, certain aspects of EGF signalling in vivo may be EGFR kinase-independent. To more fully understand the in vivo function of the EGFR kinase activity we are in the process of generating a knock-in mouse which is homozygous for the kinase-inactive form of the EGFR. In the event that the phenotype of this knock-in mouse is indistinguishable from that of the EGFR knockout mouse (i.e. early lethality), we plan to cross the F1 heterozygotic knock-in mouse with mice carrying the loxP-modified alleles of the wild type EGFR (conditional knockout).